Attention-deficit hyperactivity disorder (ADHD) is a common, neuropsychiatric disorder that affects 3-5% of children. Although the causes of ADHD are complex and not well understood, significant evidence implicates dysfunction in the brain's dopamine (DA) system. The presynaptic dopamine transporter (DAT) is a critical regulator of DA signaling and homeostasis. Heritable genetic variation in the DAT gene has been associated with ADHD, though specific functional changes have proven elusive. Recently, we (Mazei-Robison et al., 2008) described the presence of a rare DAT variant, Ala559Val (A559V), in two male siblings with ADHD. Discovery that the A559V DAT variant supports anomalous DA leak, and that this leak can be reversed by common ADHD medications has provided new opportunities to gain insight into ADHD mechanisms. In order to further advance understanding of the functional impact of the DAT A559V variant in vivo, I propose the following Specific Aims. In Aim I, I seek to establish a colony of A559V DAT knock-in mice using techniques of homologous recombination. Rigorous analysis of these mice will be pursued to insure proper integration of the variant at the mouse DAT locus, germ-line transmission, and to generate the mutation on two genetic backgrounds. In Aim II, I use biochemical and cell biological methods including DA transport assays and biochemical and immunocytochemical assessment of DAT expression to evaluate the impact of the DAT A559V variant on neurochemical aspects of the mouse brain. In Specific Aim III, adaptive changes resulting from the presence of A559V DAT will be evaluated including DA receptor expression and coupling efficiency, response of extracellular DA levels and DA clearance to amphetamine, and, collaboratively, microdialysis, and carbon-fiber amperometry. Finally, since the DAT A559V mouse is a model of ADHD, behavioral characteristics such as hyperactivity and impulsivity will be evaluated including their response to ADHD medications. Through these studies and those planned by collaborators, I pursue evidence that the DAT A559V variant can produce features expected of an ADHD model.